The present invention relates to a compound of the formula I 
its preparation and use in pairing and/or test systems.
Pairing systems are supramolecular systems of non-covalent interaction, which are distinguished by selectivity, stability and reversibility, and their properties are preferably influenced thermodynamically, i.e. by temperature, pH and concentration. DNA and RNA play a fundamental role here as carriers of the genetic traits. Such pairing systems can be used, for example, as a result of their selective properties but also as xe2x80x9cmolecular adhesivexe2x80x9d for the bringing together of different metal clusters to give cluster associations having potentially novel properties [R. L. Letsinger, et al., Nature, 1996, 382, 607-9; P. G. Schultz et al., Nature, 1996, 382, 609-11).
Strong and thermodynamically controllable pairing systems therefore play a more and more important role for use in the field of nanotechnology, for the preparation of novel materials, diagnostics, therapeutics and microelectronic, photonic and optoelectronic components and for the controlled bringing together of molecular species to give supramolecular units, such as, for example, the (combinatorial) synthesis of protein assemblies [see, for example, A. Lombardi, J. W. Bryson, W. F. DeGrado, Biomolekxc3xcls (Pept. Sci.) 1997, 40, 495-504].
For the preparation of pairing systems of this type, DNA and RNA units, however, have the following disadvantages:
a) The forces which hold together the two strands, especially hydrogen bridges and stacking effects, are naturally very low. Such duplices therefore have a low stability. This can be easily determined by recording of a so-called transition curve and determination of the transition point. Consequently, for the preparation of pairing systems relatively long individual strands are necessary, which has the result that the portion of the pairing system on the supramolecular unit predominates, i.e. the xe2x80x9cnucleotide loadxe2x80x9d is high.
b) Owing to the formation of Hoogsteen pairings, which are possible alternatively to Watson-Crick pairings, the selectivity decreases. Parallel duplices or irreversible pairing processes are thus often combined.
c) Owing to the high flexibility of the sugar phosphate backbone, helical conformations are formed, as a result of which the spatial arrangement in supramolecular units can be less readily controlled.
d) The chemical instability of the phosphodiester bond in the backbone permits only a slight variance in physical conditions, such as pH or temperature, for the use of the supramolecular units.
e) The nuclease sensitivity of the products leads to a rapid enzymatic degradation, which can only be avoided with difficulty, and thus to the destruction of the supramolecular unit.
f) Possible interference with the genetic material of biological systems is not to be excluded if the supramolecular units are used in a biological system, i.e. an orthogonality of the pairing process is absent.
g) The preparation of relatively large amounts of oligonucleotides is difficult on account of the low loading ability of the solid phase customarily used, for example in comparison with peptide synthesis.
h) The preparation of the unnatural L enantiomeric form is made difficult by the poor accessibility of the appropriately configured sugar units.
Thus use of DNA or RNA units, for example, in complementarily bonded two- and three-dimensional supramolecular structures (see, for example, WO96/13522) in a physiological medium can only be realized with difficulty, especially in view of item e), f) and g).
An alternative to DNA and RNA units is the so-called pyranosyl-RNA (p-RNA). pRNA is an oligonucleotide which contains ribopyranose as a sugar unit instead of ribofuranose and therefore exclusively forms Watson-Crick-paired, antiparallel, reversibly xe2x80x9cmeltingxe2x80x9d, quasi-linear and stable duplices. In addition, there are also homochiral p-RNA strands with an opposite sense of chirality, which likewise pair controllably and are not strictly helical in the duplex formed. This valuable specificity for the synthesis of supramolecular units is connected with the relatively low flexibility of the ribopyranose phosphate backbone and with the strong inclination of the base plane to the strand axis and the tendency resulting from this for intercatenary base stacking in the resulting duplex and can finally be attributed to the participation of a 2xe2x80x2,4xe2x80x2-cis-disubstituted ribopyranose ring in the synthesis of the backbone. p-RNA thus solves some of the described disadvantages of DNA and RNA, but not the disadvantages according to items d), e), g) and h).
A further alternative is the linkage of the monomer units by means of amide bonds, i.e. the synthesis of an oligomeric peptide, so-called peptide nucleic acids (PNAs).
Owing to their open-chain structure, PNAs have a high flexibility and are thus not suitable for the controlled synthesis of supramolecular systems in view of their conformational preorganization.
It is therefore the object of the present invention to make available compounds which do not have the disadvantages described above.
One subject of the present invention is therefore compounds of the formula I 
in which R1 is equal to NR3R4, OR3 or SR3 where R3 and R4 independently of one another, identically or differently, are H or CnH2n+1, n being an integer from 1-12, preferably 1-8, in particular 1-4; preferably R1 is equal to NR3R4 or OR3, in particular NR3R4, especially NH2;
R2 is equal to CmH2mxe2x80x94C(X)xe2x80x94Y where X is equal to xe2x95x90O, xe2x95x90S or xe2x95x90Nxe2x80x94H, wherein Y is OR3, NR3R4 or SR3, or R2 is equal to CmH2mxe2x80x94CH2xe2x80x94X or CmH2mxe2x80x94CH(X)xe2x80x94Y, wherein X is equal to NR3R4, OR3, or SR3; or R2 is equal to CmH2mxe2x80x94Zxe2x80x94Yxe2x80x2 where Z is equal to a S(xe2x95x90O)2 or P(xe2x95x90O)(Oxe2x88x92), wherein Yxe2x80x2 is equal to H, CnH2n+1, OR3, NR3R4 or SR3, and wherein m is an integer from 1-4;
A, B and D independently of one another, identically or differently, are CR5R6, O, NR7 or S where R5, R6, R7 independently of one another are H or CnH2n+1, where n has the abovementioned meaning; and
C is equal to CR8 or N, where R8 independently thereof denotes the meaning of R5, but where Axe2x80x94B, Bxe2x80x94C or Cxe2x80x94D are not two identical heteroatoms; and
the term nucleobase within the meaning of the present invention denotes thymine, uracil, adenine, cytosine, guanine, isocytosine, isoguanine, xanthine or hypoxanthine, preferably thymine, uracil, adenine, cytosine or guanine.
In particular, compounds are preferred in which R1 is equal to NH2 and R2 is equal to CH2xe2x80x94COOH, especially a [2-amino-4-(carboxymethyl)cyclohexyl]nucleobase, such as 1-[2-amino-4-(carboxymethyl)cyclohexyl]thymine, 1-[2-amino-4-(carboxymethyl)cyclohexyl]uracil, 1-[2-amino-4-(carboxymethyl)cyclohexyl]-cytosine, 9-[2-amino-4-(carboxymethyl)cyclohexyl]adenine or 9-[2-amino-4-(carboxymethyl)cyclohexyl]guanine.
It is furthermore advantageous if the compound according to the invention is enantiomerically pure.
For the synthesis, it is further advantageous if R1 is also provided with protective groups, such as, for example, in the case of R1 is equal to NH2 with tert-butoxycarbonyl groups (BOC) or 9-fluorenylmethoxycarbonyl groups (FMOC) or in the case of R1 equal to OH, with ether or acetal protective groups. Protective groups are in general understood as meaning radicals which protect reactive groups of compounds from undesired reactions and are easily removable. Groups of this type are, for example, benzyloxymethyl (BOM), BOC, FMOC, ether or acetal protective groups.
In addition, Y can also be reacted with activating reagents to give reactive intermediates, e.g. mixed anhydrides.
Preferably, the compounds according to the invention are cyclohexane derivatives which, in the 1xe2x80x2-position, carry a nucleobase and in the 2xe2x80x2-position a nucleophile, such as, for example, a nitrogen atom which can be reacted with the reactive group in the 4xe2x80x2-position or after its activation, such as, for example, with a carbonyl function, and thus by repetition of this process is able to build up an oligomeric structure. From the stereochemical point of view, derivatives are preferred in which all substituents on the 6-membered ring have equatorial positions, in particular in which the substituents in the 1xe2x80x2- and 2xe2x80x2-position have equatorial positions and especially in which the nucleobase has an equatorial position.
The compounds according to the invention can be prepared, for example, in the following way.
In the beginning, a chirogenic Diels-Alder reaction is carried out (see FIG. 1) between, for example, 1,3-butadiene 1 and, for example, 3-(2-propenoyl)-1,3-oxazolidin-2-one 2 in the presence of chiral, non-racemic promoters such as the Seebach""s TADDOL 3 (D. Seebach et al., J. Org. Chem. 1995, 60, 1788). 3-(2-Propenoyl)-1,3-oxazolidin-2-one can be prepared, for example, by reaction of oxazolidin-2-one with acryloyl chloride in the presence of copper and copper(I) chloride. The compound 5 can be prepared, for example, from 4 in the presence of magnesium turnings and anhydrous methanol. The reaction product is then reduced, for example, with lithium aluminium hydride in order to give the compound 6. The acetonitrile 7 can be prepared from 6, for example, by reacting with methanoyl chloride to give methyl methanoate and then by reacting with cyanide. Alkaline hydrolysis of the reaction product forms the acetic acid derivative, which can be reacted with SOCl2 to give the acetyl chloride to afford the acetamide 8 in the presence of an aqueous ammonia solution. Iodolactamization thereof is then carried out (S. Knapp et al., Org. Synth. 1991, 70, 101) to give the iodolactam 9.
The iodolactam 9 can then be coupled to a nucleobase in the presence of a hydride without the bicyclic system being destroyed.
For this, it is advantageous if possibly reactive groups of the nucleobase are protected by suitable protective groups, e.g. BOC, FMOC, acetal etc. Only in the case of diaminopurine as a nucleobase are protective groups unnecessary. However, it has been shown that in the case of diaminopurine as a nucleobase it was no longer possible to open the lactam ring. Independently of this, diaminopurine plays no role in biological systems.
It is furthermore preferred if the iodolactam is not present in racemic form, but in enantiomerically pure form, as the oligomers to be synthesized, which are intended to be a pairing system, should belong only to one stereochemical series. The synthesis of a pairing system from racemic units is in general not possible.
According to the present invention, a couplable pyrimidine unit, e.g. a thymine unit, can be prepared starting from the iodolactam 9 in a five-stage synthesis sequence in a yield of 27% over all steps (FIG. 2.):
In the first step, the stereoselective introduction of the nucleobase is carried out in a substitution reaction (13a) followed by the masking of the acidic imide proton of the thymine (13b). The lactam is then activated by introducing a protective group, e.g. the Boc group (13c), and the ring is opened nucleophilically, for example, by means of LiOOH (14a). After removing the BOM protective group which, for example, is present, the desired unit (14b) results by means of catalytic hydrogenation.
The enantiomorphic (R) unit ent-14b is obtainable by an analogous procedure from the (R)-iodolactam ent-9. Likewise, couplable uracil, cytosine and isocytosine units are obtainable analogously.
A couplable purine unit, e.g. an adenine unit, is accessible starting from the iodolactam 9 in a seven-stage synthesis sequence in a yield of 19% over all steps (FIG. 3.):
After introduction of the, for example, N(6)-benzoylated adenine into the (S)-iodolactam 9 with retention (15a) the reprotection to give the, for example, N(6)-formamidineadenine lactam 15c is carried out. The activation of the lactam, for example, by means of tert-butoxycarbonylation (15d), a new reprotection to give the, for example, N(6)-anisoyl-protected lactam (15f) and subsequent lactam cleavage, for example, by means of LiOH yields the desired unit 16 are then carried out.
The enantiomorphic (R) unit ent-16 is obtainable by an analogous procedure from the (R)-iodolactam ent-9. Likewise, couplable guanine, isoguanine, xanthine, and hypoxanthine units are obtainable analogously.
The preparation of heteroatom-containing six-membered rings and other derivatives of the compound according to the invention are [sic] possible, for example, analogously by means of a hetero-Diels-Alder reaction or by means of Diels-Alder reactions with the appropriate starting compounds.
Another subject of the present invention is therefore a process for the preparation of a compound according to the invention having the following steps:
(a) coupling of the appropriate iodocycloalkane, e.g. of an iodolactam, preferably of an enantiomerically pure iodocycloalkane, with a protected nucleobase preferably in the presence of a hydride, such as, for example, NaH, and in the case of an iodolactam
(b) activation of the lactam, for example by introducing a protective group such as, for example, a BOC group, and
(c) nucleophilic ring-opening, e.g. by means of a hydroperoxide such as, for example, LiOOH.
For the preparation of oligomers, the compounds according to the invention are then preferably synthesized on a solid phase according to the Merrifield peptide synthesis to give oligomeric structures. In this process, the necessary reagents are preferably added in an excess and unreacted amounts are removed again by simple washing steps [R. B. Merrifield, J. Amer. Chem. Soc. 1963, 85, 2149]. Repetitive cycles which consist of removals of the temporary protective groups and couplings, e.g. according to the symmetrical anhydride method, of the protected monomer units lead to the oligomers on the resin. At the end of the synthesis, the terminal protective group is removed, and the oligomer is cleaved from the resin and preferably purified using chromatographic methods. The isolated HPLC fractions of the synthesis products were checked for their purity using analytical HPLC and clearly characterized using electrospray mass spectrometry.
A further subject of the present invention is therefore an oligomer, called CAN (cyclohexylnucleooligoamide), comprising at least one compound according to the invention, which in general are [sic] linked 2xe2x80x2xe2x86x924xe2x80x2.
In order to bring together molecular species, such as peptides, proteins, receptors, redox centres, antibodies, nucleic acid sections (such as DNA, RNA) with the aid of the pairing system described, in general suitable linkers are integrated. Preferably, but not exclusively, lysine can be incorporated at any desired position in the oligomeric pairing system, but very preferably terminally. Owing to its free amino group, lysine has a large number of linkage possibilities. Incorporation into the oligomer according to the invention is carried out, for example, by means of Boc-Lysine(Fmoc), whose FMOC group can be removed using piperidine in DMF.
The present invention therefore also relates to oligomers which additionally contain at least one linker, preferably a lysine linker, especially a terminal lysine linker.
The, for example, amino-functionalized oligomer can also preferably be derivatized with activated linkers such as, for example, iodoacetylsuccinimide (A) or bis(hydroxysuccinimidyl)glutarate (B), which then preferably react in physiological media, in particular in aqueous-buffered solutions optionally with addition of organic solubilizers, with HS groups of the molecular species, e.g. via (A) with the cysteine radicals of a peptide or protein or via (B) with amino groups of the molecular species with formation of chemically stable, covalent forms.
The present invention therefore also includes oligomers which are derivatized with an activated linker, such as, for example, iodoacetylsuccinimide and/or bis(hydroxysuccinimidyl)glutarate.
The oligomers according to the invention having a free N and C terminus are generally only poorly soluble in water at pH 7. In the case of tetramers, concentrations in the 100 xcexcmolar, in the case of pentamers in the 10 xcexcmolar and in the case of hexamers only approximately in the 1 xcexcmolar range, are achieved. These are approximate values which, depending on the sequence, can also be exceeded or fallen short of by an order of magnitude. The solubility of AAATT is, for example, 1 xcexcM, that for AATAT 50 xcexcM.
By acylation of the N terminus with a hydroxycarboxylic acid derivative, it is possible, for example, to introduce a hydroxyl function which can be phosphorylated. The phosphated oligomer thus obtained customarily is at least about 1000 times more soluble in water at about pH 7. The solubility is generally detected by UV spectroscopy. The present invention therefore also relates to phosphated oligomers.
Another subject of the invention is also a conjugate of an oligomer according to the invention and a biomolecule.
Biomolecule is understood according to the present invention as meaning, for example, a peptide, peptoid or protein, such as, for example, a receptor, an antibody or functional parts thereof or an enzyme, and a nucleic acid such as DNA or RNA, or cell constituents such as lipids, glycoproteins, filament constituents, or viruses, virus constituents such as capsids, viroids, and their derivatives such as, for example, acetates.
Functional parts of antibodies are, for example, Fv fragment (Skerra and Plxc3xcckthun (1988) Science 240, 1038), single-chain Fv fragments (scFv; Bird et al (1988), Science 242, 423; Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A., 85, 5879) or Fab fragments (Better et al. (1988) Science 240, 1041). The conjugates according to the invention of effector molecules and preferably peptide, but in contrast to PNA, selective and controllable pairing systems are advantageous if reversibly supramolecular assemblies are to be synthesized, whose action, such as, for example, binding, inhibition, induction of a physiological effect, differs from the action of the individual effector molecules.
Thus, for example, WO 96/13613 describes a method for finding a substance which induces a biological action due to the multimerization of proteins by first determining a substance I which binds to a protein by means of a test, then determining a second substance II which binds to a second protein and then covalently linking the two substances I and II by means of a linker such that a dimerization of the two proteins is induced. This dimerization then brings about the desired biological effect. Such a method can achieve greater flexibility if the linkage of the two substances I and II takes place non-covalently, but by a pairing system such as the oligomer or conjugate according to the invention. Owing to the controllability of this pairing, for example by temperature or pH, the dimerization process of the proteins can be observed or its extent controlled. The pairing systems according to the invention have the advantage, for example compared with the systems from WO 96/13522, that they are nuclease-stable.
An attempt to use peptide xe2x80x9cadhesivexe2x80x9d units for the formation of homo- or heterodimeric assemblies is described, for example, in WO 94/28173:
Association peptides (hexa- or heptapeptides) of a fixed sequence bring together effector units, such as, for example, proteins, to give supramolecular units. Such a method can achieve higher flexibility due to controllability of this association process, which in general cannot be realized with the association peptides, but advantageously with the pairing systems of the present invention.
The invention therefore also relates to the use of the compound according to the invention, the oligomer according to the invention or the conjugate according to the invention in a pairing and/or test system, such as, for example, described in greater detail in WO 94/28173, WO 96/13522, WO 96/13613, R. L. Letsinger, et al., Nature, 1996, 382, 607-9; P. G. Schultz et al., Nature, 1996, 382, 609-11 or A. Lombardi, J. W. Bryson, W. F. DeGrado, Biomolekxc3xcls (Pept. Sci.) 1997, 40, 495-504 and generally explained above.
A particular embodiment of the present invention is a carrier on which a compound according to the invention, an oligomer according to the invention and/or a conjugate according to the invention is immobilized, in particular for use in a pairing and/or test system, as described in greater detail above.
The term xe2x80x9cimmobilizedxe2x80x9d is understood within the meaning of the present invention as meaning the formation of a covalent bond, quasi-covalent bond or supramolecular bond by association of two or more molecular species such as molecules of linear constitution, in particular peptides, peptoids, proteins, linear oligo- or polysaccharides, nucleic acids and their analogues, or monomers such as heterocycles, in particular nitrogen heterocycles, or molecules of non-linear constitution such as branched oligo- or polysaccharides or antibodies and their functional moieties such as Fv fragments, single-chain Fv fragments (scFv) or Fab fragments.
Suitable carrier materials are, for example, ceramic, metal, in particular noble metal, glasses, plastics, crystalline materials or thin layers of the carrier, in particular of the materials mentioned, or (bio)molecular filaments such as cellulose, structural proteins.
In summary, the present invention has the following advantages compared with conventional pairing and test systems:
The duplices of the oligomers or conjugates according to the invention are significantly more stable than those of DNA, RNA and p-RNA. The oligomeric substances are significantly more stable chemically and against enzymatic degradation, do not pair with DNA or RNA and can be prepared in relatively large amounts in a simple manner. Both enantiomers are easily accessible by synthetic chirogenic steps. The compounds according to the invention are therefore superior to the pairing systems known hitherto as selective xe2x80x9cmolecular adhesivesxe2x80x9d.
The following figures and examples are intended to describe the invention in greater detail without restricting it.